CN117004553A - Woody musk glandular cell culture medium and preparation method and application thereof - Google Patents

Woody musk glandular cell culture medium and preparation method and application thereof Download PDF

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CN117004553A
CN117004553A CN202310998679.7A CN202310998679A CN117004553A CN 117004553 A CN117004553 A CN 117004553A CN 202310998679 A CN202310998679 A CN 202310998679A CN 117004553 A CN117004553 A CN 117004553A
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culture medium
musk
cells
cell culture
woody
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姚丛学
廖丹丹
安娴
夏传英
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Beijing Yanshe Biotechnology Development Co ltd
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Abstract

The application relates to the technical field of cell culture, and particularly discloses a woody musk glandular cell culture medium and a preparation method and application thereof. The application discloses a culture medium, which takes DMEM/F12 as a basic culture medium; comprising as functional additives the following concentrations of components: penicillin 80-120U/ml, streptomycin 0.08-0.12mg/ml, FBS 8-12v/v%, optional amino acid 0.7-1.3v/v%, growth factor 20-40ng/ml, and vitamin 0.05-0.2 μg/ml. The application also discloses a preparation method of the culture medium and application of the culture medium in culture of woody musk glandular cells. When the culture medium disclosed by the application is used for culturing the musk gland cells, the cell proliferation speed of the Lin Shexiang gland cells is higher, the cell activity is stronger, and the cell adherence rate is higher.

Description

Woody musk glandular cell culture medium and preparation method and application thereof
Technical Field
The application relates to the technical field of cell culture, in particular to a woody musk glandular cell culture medium and a preparation method and application thereof.
Background
Forest musk deer is a precious animal, and fragrance of forest musk deer is widely applied to chemical and medical fields such as spice, medicine and the like. However, musk resources are in short supply due to the scarcity of forest musks and the extremely low individual musk yields.
Cell culture technique the cell culture technique refers to a method of simulating in vitro the in vivo environment (sterility, proper temperature, pH value, certain nutrient conditions, etc.) to survive, grow, reproduce and maintain the main structure and function. Cell culture techniques refer to the growth of cells under in vitro conditions, during which the culture is a single cell or a population of cells. The cells are all living in an artificial environment during culture, and the cells move or are influenced by other factors due to the change of the environment, so that the culture time is prolonged, and the cells are subjected to passage to form a single type. Moreover, cell culture itself is a cloning of cells, and cell culture techniques are important and common techniques in cell biology research methods. The cell culture can be used for obtaining a large number of cells and researching molecular mechanisms such as signal transduction of the cells, anabolism of the cells and the like.
However, since Lin Shexiang gland cells are precious and difficult to collect, the requirement for cell culture media is high, so that the research on forest musk deer by using cell culture technology is relatively few. However, the cell culture techniques commonly used at present are not suitable for the long-term growth and propagation of Lin Shexiang glandular cells, and seriously hamper the research on forest musk deer.
Disclosure of Invention
In order to solve the technical problems in the prior art, the application provides a woody musk glandular cell culture medium and a preparation method and application thereof.
The application provides a culture medium, which takes DMEM/F12 as a basic culture medium;
the medium contained the following concentrations of components as functional additives: penicillin 80-120U/ml, streptomycin 0.08-0.12mg/ml, FBS 8-12v/v%, optional amino acid 0.7-1.3v/v%, growth factor 20-40ng/ml, vitamin 0.05-0.2 μg/ml; the growth factor is selected from one or more of EGF, FGF, IL-1.
The application uses the components with the concentrations as functional additives, the used unnecessary amino acid is Gibco finished product, gibco is a common unnecessary amino acid, the ratio of cell culture medium can be effectively improved, and the side effect of cell self production on the unnecessary amino acid during cell culture is reduced; the growth factors can improve the survival rate, promote the growth and division of cells, and promote the proliferation of the cells; the vitamins can maintain the normal growth and development of cells and promote the proliferation of cells. The application obtains the culture medium for culturing musk gland cells by adjusting the concentration of each component, and can promote the growth and the adherence of cells.
Preferably, the growth factor is (7-11) by weight: EGF, IL-1 of (1-3).
EGF is also called as human oligopeptide-1, which can promote cell proliferation and differentiation and increase cell renewal rate; FGF is an important morphogenesis and differentiation inducer, has the effects of promoting vascular growth, promoting wound healing and tissue repair, participating in nerve regeneration and the like, and can play a role in vitro at lower concentration; by adopting the growth factors with the proportion, the division and the update of the mesenchymal stem cells can be effectively promoted, and the culture speed and the culture quality are improved.
In a specific embodiment, the FGF, IL-1 may be present in a weight ratio of 7: 1. 7: 2. 7: 3. 9: 1. 9: 2. 9: 3. 11: 1. 11: 2. 11:3.
in some specific embodiments, the weight ratio of FGF to IL-1 can also be (7-9): 1. (7-9): 2. (7-9): 3. (9-11): 1. (9-11): 2. (9-11): 3. 11: (1-2), 11: (2-3), 11: (1-3).
Experimental analysis shows that the culture medium is used for culturing the musk gland cells by selecting the FGF and the IL-1 with the weight ratio and adding the FGF and the IL-1 into the culture medium, so that the culture effect of the musk gland cells can be further improved.
Preferably, the vitamin is selected from one or more of vitamin C, vitamin B12, vitamin H.
Preferably, the medium further comprises trace elements in the range of 0.001-0.04. Mu.g/ml.
Preferably, the microelements are selected from one or more of ferric ammonium citrate, sodium selenate and sodium selenite.
Further, the microelements comprise the following components in parts by weight (2-6): (0.02-0.08) ferric ammonium citrate and sodium selenate.
In a specific embodiment, the weight ratio of ferric ammonium citrate to sodium selenate may be 2:0.02, 2:0.05, 2:0.08, 4:0.02, 4:0.05, 4:0.08, 6:0.02, 6:0.05, 6:0.08.
in some specific embodiments, the weight ratio of ferric ammonium citrate to sodium selenate may also be (2-4): 0.02, (2-4): 0.05, (2-4): 0.08, (4-6): 0.02, (4-6): 0.05, (4-6): 0.08, 6: (0.02-0.05), 6: (0.05-0.08), 6: (0.02-0.08).
Experimental analysis shows that the application selects ferric ammonium citrate and sodium selenate with the weight ratio as microelements to be added into a culture medium, and the culture medium is used for culturing the musk gland cells, so that the culture effect of the musk gland cells can be further improved.
In a second aspect, the application also provides a preparation method of the Lin Shexiang glandular cell culture medium, which specifically comprises the following steps:
weighing corresponding raw materials, fully dissolving in 950mL of basic culture medium DMEM/F12, adjusting pH to 6.6-7.2, adding sterile deionized water to 1000mL, and filtering with a filter membrane with aperture of 0.22 μm to obtain culture medium, and preserving at 4deg.C for use.
In a third aspect, the application also provides application of the Lin Shexiang glandular cell culture medium in culture of musk glandular cells.
In a fourth aspect, the application provides a method for culturing musk gland cells in a forest, which comprises the following steps: in the culture medium according to the ratio of (0.5-5) x 10 3 The cells/mL inoculation density was used to inoculate woody musk adeno cells, then at 37℃with 5% CO 2 Is cultured under saturated humidity condition.
In summary, the technical scheme of the application has the following effects:
the application takes DMEM/F12 as a basic culture medium, takes penicillin, streptomycin, FBS, unnecessary amino acid, growth factors and vitamins as functional additives, and then prepares the culture medium for culturing the musk gland cells, which can promote cell growth and cell adhesion, thereby obtaining the musk gland cells with high cell density, high cell activity and high adhesion rate.
The application further improves the culture effect of the culture medium on the woody musk gland cells by screening the types of the growth factors and the vitamins and adding microelements with proper types and dosage into the culture medium.
Drawings
FIG. 1 is a microscopic view (10X) of example 7 at 0h of the inoculation of Lin Shexiang glandular cells.
FIG. 2 is a microscopic view (10X) of comparative example 6 at 0h of woody musk glandular cell inoculation.
FIG. 3 is a microscopic view (10X) of comparative example 7 at 0h of Lin Shexiang glandular cell inoculation.
FIG. 4 is a microscopic view (10X) of example 7 at 4d of the culture of Lin Shexiang glandular cells.
FIG. 5 is a microscopic view (10X) of comparative example 6 at 4d of the time of the culture of Lin Shexiang glandular cells.
FIG. 6 is a microscopic view (10X) of comparative example 7 at 4d of the time of the culture of Lin Shexiang glandular cells.
Detailed Description
The present application is described in further detail below in connection with examples, comparative examples, and performance test runs, which are not to be construed as being included within the scope of the claimed application.
Preparation example
Preparation examples 1 to 12
Preparation examples 1 to 12 each provide a medium.
The preparation examples are different in that: composition of growth factors and concentration of growth factors in the medium. Specifically, the results are shown in Table 1.
The preparation method of the culture medium comprises the following steps:
weighing raw materials according to the final concentration of penicillin 100U/ml, streptomycin 0.1mg/ml, FBS10v/v%, optional amino acid (Gibco) 1v/v% and vitamin H0.1 μg/ml according to 1L standard culture medium; then according to Table 1, weighing the growth factors with corresponding weight, fully dissolving in 950mL of basic culture medium DMEM/F12, regulating pH to 6.6-7.2, adding sterile deionized water to 1000mL, fixing the volume, filtering with a filter membrane with the pore diameter of 0.22 μm to obtain a culture medium, and preserving at 4 ℃ for later use.
TABLE 1 types of growth factors and addition amounts of growth factors in preparation examples 1 to 12
Preparation examples 13 to 14
Preparation examples 13 to 14 each provide a medium.
Each of the above preparation examples is different from preparation example 8 in that: the types of vitamins in the culture medium are as follows:
the vitamin used in the culture medium in preparation example 13 is vitamin C;
the vitamin used in the medium of preparation example 14 was vitamin B12.
Preparation examples 15 to 29
Preparation examples 15 to 29 each provide a medium.
Each of the above preparation examples is different from preparation example 8 in that: the culture medium also comprises trace elements, and the composition and concentration of the trace elements are shown in table 2.
TABLE 2 compositions and concentrations of microelements in preparation examples 8, 15-29
PREPARATION EXAMPLES 30 to 33
Preparation examples 30-34 each provide a medium.
The above preparation examples and preparation example 8 are different as follows:
the concentration of growth factor in the medium of preparation example 30 was 10ng/ml;
the concentration of growth factor in the medium of preparation 31 was 55ng/ml;
the growth factor in the medium of preparation 32 was PDGF.
The medium in preparation 33 does not contain vitamins.
The medium in preparation 34 does not contain nonessential amino acids.
Preparation example 35
Preparation 35 provides a medium.
The preparation method of the culture medium in the preparation example comprises the following steps:
weighing raw materials according to the standard 1L culture medium, namely penicillin-streptomycin, FBS20v/v%, EGF 10ng/mL, glutamine 2mM and Mbeta-mercaptoethanol 0.1mM with the final concentration of 1%, fully dissolving the raw materials in 950mL of basal culture medium Ham's F12 culture medium, regulating pH to 6.6-7.2, adding sterile deionized water to 1000mL, and filtering with a filter membrane with the aperture of 0.22 mu M to obtain the culture medium, and preserving at 4 ℃ for later use.
Preparation example 36
Preparation 36 provides a medium.
The preparation method of the culture medium in the preparation example comprises the following steps:
weighing raw materials according to a standard of 1L of culture medium, wherein the final concentration of the raw materials is 0.5mg/mL of bovine insulin, 0.5mg/mL of casein, 1.5 mu g/mL of transferrin, 15 mu g/mL of bovine insulin, 5 mu g/mL of linoleic acid, 400nmol/L of testosterone, 0.15mmol/L of vitamin A, 0.05mol/L of L-ascorbic acid, 0.26mmol/L of polylysine, 1nmol/L of putrescine and 25 mu mol/L of 2-mercaptoethanol;
the raw materials are fully dissolved in 950mL of basic culture medium DMEM/F12 respectively, the pH is regulated to 6.6-7.2, sterile deionized water is added to fix the volume to 1000mL, and then a filter membrane with the aperture of 0.22 mu m is used for filtering, thus obtaining the culture medium, and the culture medium is preserved at the temperature of 4 ℃ for standby.
Examples
Examples 1 to 29
Examples 1-29 provide a method for culturing woody musk gland cells, respectively.
The above-described embodiments differ in that: the media used in Lin Shexiang glandular cell culture were different, and the media used in examples 1 to 29 were derived from preparation examples 1 to 29, respectively.
The method for culturing the musk gland cells in the embodiment specifically comprises the following steps:
isolation of cells from the thymus tissue of woody musk deer: after anesthetizing forest musk using zolazepam hydrochloride, let musk lie flat and cut the skin above musk gland with ophthalmic scissors to obtain gland tissue. The removed sample tissue was immediately transferred to physiological saline containing 100U/ml penicillin and 100. Mu.g/ml streptomycin.
Digestion: the sample tissue was minced to about 1mm with sterile ophthalmic scissors, and digested with 0.25mg/mL trypsin containing EDTA for about 3-10min.
Cell preparation: digestion was stopped with 100IU/ml penicillin-streptomycin in phosphate buffer (PBS for short), pipetted into a centrifuge tube, centrifuged at 1500r/min for 5min, the supernatant discarded, the resulting cell suspension added to complete medium and centrifuged again, repeated 1 time, and the dispersed cells were collected.
Cell culture: diluting the prepared single cell solution to 3×10 4 cells/mL, then inoculated in 25T cell culture flasks at 37℃with 5% CO 2 Culturing in a saturated humidity incubator, changing the complete culture medium after the cells are attached, and after about 5 days, the cells are migrated, and then changing the culture medium every 48 hours.
Comparative example
Comparative examples 1 to 7
Comparative examples 1-7 provide a method of culturing woody musk gland cells, respectively.
The above comparative examples are different in that: the culture media used in the Lin Shexiang glandular cell culture method were different, and the cultures used in comparative examples 1-7 were derived from preparation examples 30-36, respectively.
Performance test
The cell density, cell activity and attachment rate of cells in the culture process of Lin Shexiang glandular cells were measured by the methods of examples 1 to 29 and comparative examples 1 to 7.
The detection method comprises the following steps: taking a part of cells every day after 1d, 2d, 4d and 8d after the culture of the musk gland cells, taking supernatant after digestion of 0.25% pancreatin containing EDTA, counting the cells by using a blood cell counting plate, and determining the cell density of the cells; determining the cell activity of the cells by trypan blue staining; the attachment rate of the cells was examined by microscopic visual observation.
Detection result:
1-5 are microscopic views of woody musk gland cells, wherein FIG. 1 is a microscopic view (10 x) of example 7 at 0h of inoculation of Lin Shexiang gland cells, FIG. 2 is a microscopic view (10 x) of comparative example 6 at 0h of inoculation of woody musk gland cells, FIG. 3 is a microscopic view (10 x) of comparative example 7 at 0h of inoculation of Lin Shexiang gland cells, FIG. 4 is a microscopic view (10 x) of example 7 at 4d of inoculation of Lin Shexiang gland cells, FIG. 5 is a microscopic view (10 x) of comparative example 6 at 4d of inoculation of Lin Shexiang gland cells, and FIG. 6 is a microscopic view (10 x) of comparative example 7 at 4d of inoculation of Lin Shexiang gland cells;
the cell densities, cell activities and attachment rates of the musk gland cells of examples 1 to 29 and comparative examples 1 to 7 are shown in Table 3.
TABLE 3 cell Density, cell Activity and attachment Rate of Adenocyte of Lin musk in examples 1-29 and comparative examples 1-7
In combination with Table 3, it is understood that the culture medium prepared by the present application is used for culturing the woody musk gland cells according to the results of the cell density, cell activity and adherence of the woody musk gland cells of comparative examples 1 to 29 and comparative examples 1 to 7, and thus the woody musk gland cells with high cell density, high cell activity and high adherence can be obtained.
Referring to fig. 3, 6 and table 3, the medium used in comparative example 7 is a conventional medium used for culturing woody musk gland cells, and the attachment rate of the woody musk gland cells after 4d culture is as low as 0, so that the cells cannot grow and most of the cells die; the cell density of the woody musk gland cells after 8d culture is as low as 0.1 multiplied by 10 4 cell/mL, cell activity was as low as 21.8%.
In combination with FIGS. 2, 5 and Table 3, the culture medium used in comparative example 6 was used for culturing woody musk gland cells, and the culture medium was inferior in cell density, cell activity and attachment rate of Lin Shexiang gland cells.
Comparative example 4 does not use vitamins as functional additives, and comparative example 5 does not use nonessential amino acids as functional additives, and the culture medium prepared in this way has poor cell culture effect when used for culturing woody musk gland cells.
From the results of comparison of examples 1 to 12 with comparative examples 1 to 3, it was found that the culture effect of the woody musk gland cells was deteriorated by too low a concentration of the growth factor or too high a concentration of the growth factor in the culture medium in comparative examples 1 to 3 or by using other kinds of growth factors. Thus, the present application chooses to use growth factors in the medium at a concentration of 20-40 ng/ml. Further, according to the results of comparative examples 1 to 10, the present application was conducted with the weight ratio of (7 to 11): EGF and IL-1 of (1-3) are used as growth factors, and the culture effect of the musk gland cells can be further improved.
From the results of comparison of examples 8 and 13 to 14, it was found that the culture effect of Lin Shexiang glandular cells was improved by adding vitamin H to the medium in the present application, compared to the selection of vitamin C and vitamin B12 to be added to the medium.
From the results of comparison of examples 8 and 15-29, it is apparent that the culture effect of Lin Shexiang gland cells is better improved by using one or more of ferric citrate, sodium ammonium selenate and sodium selenite as trace elements to be added into the culture medium, compared with the method of selecting copper sulfate pentahydrate or zinc sulfate heptahydrate to be added into the culture medium as trace elements. Further, the weight ratio of the selected components is (2-6): (0.02-0.08) ferric ammonium citrate and sodium selenate as microelements are added into the culture medium.
While the application has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the application and are intended to be within the scope of the application as claimed.

Claims (9)

1. A musk gland cell culture medium, which is characterized in that the culture medium takes DMEM/F12 as a basic culture medium;
the medium contained the following concentrations of components as functional additives: penicillin 80-120U/ml, streptomycin 0.08-0.12mg/ml, FBS 8-12v/v%, optional amino acid 0.7-1.3v/v%, growth factor 20-40ng/ml, vitamin 0.05-0.2 μg/ml; the growth factor is selected from one or more of EGF, FGF, IL-1.
2. The woody musk glandular cell culture medium according to claim 1, wherein the growth factors are in a weight ratio of (7-11): EGF, IL-1 of (1-3).
3. The woody musk glandular cell culture medium according to claim 1, wherein the vitamin is one or more selected from the group consisting of vitamin C, vitamin B12, vitamin H.
4. The woody musk glandular cell culture medium according to claim 1, further comprising trace elements of 0.001-0.04 μg/ml.
5. The woody musk glandular cell culture medium according to claim 4, wherein the trace elements are one or more selected from ferric ammonium citrate, sodium selenate and sodium selenite.
6. The woody musk glandular cell culture medium according to claim 5, wherein the trace elements include (2-6) in weight ratio: (0.02-0.08) ferric ammonium citrate and sodium selenate.
7. A method for preparing a Lin Shexiang glandular cell culture medium according to any one of claims 1 to 6, comprising the specific steps of:
weighing corresponding raw materials, fully dissolving in 950mL of basic culture medium DMEM/F12, adjusting pH to 6.6-7.2, adding sterile deionized water to 1000mL, and filtering with a filter membrane with aperture of 0.22 μm to obtain culture medium, and preserving at 4deg.C for use.
8. Use of a woody musk gland cell culture medium according to any one of claims 1 to 6 for culturing woody musk gland cells.
9. A method of culturing woody musk gland cells, characterized by using the culture medium according to any one of claims 1-6, comprising the following steps: in the culture medium according to the ratio of (0.4-4) x 10 5 The cells/mL inoculation density was used to inoculate woody musk adeno cells, then at 37℃with 5% CO 2 Is cultured under saturated humidity condition.
CN202310998679.7A 2023-08-09 2023-08-09 Woody musk glandular cell culture medium and preparation method and application thereof Pending CN117004553A (en)

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